Process for producing an antigen for use in the diagnosis of malaria and product thereof



Patented May 10, 1949 PROCESS FOR PRODUCING AN ANTIGEN FOR USE IN THEDIAGNOSIS OF MALARIA AND PRODUCT THEREOF Anna Dean Dulaney, Memphis,Tenn., assignor to The University of Tennessee Research Corporation,Knoxville, Tenn., a corporation of Tennessee No Drawing. Originalapplication September 17,

1945, Serial No. 616,959. Divided and this application December 10,1948, Serial No. 64,696

Claims.

This invention relates to the production of an improved antigen for usein the diagnosis of malaria.

Complement fixation tests have been developed to aid in routinelaboratory determination of malaria. They depend upon the use ofantigens ordinarily produced as saline extracts in known manner fromdried parasites ground with saline solution and successively frozen,thawed and centrifuged '(Dulaney and Stratman-Thomas, J. Immun. 1940,vol. 39, pp. 247-255). While this procedure has proven dependable forsuch tests, yet the opinion is justified that the test is more specificthan sensitive. Recognition of the diagnostic possibilities of the testemphasizes the need for a more sensitive antigen.

It is an object of the present invention to produce a highly activeantigen for the diagnosis of malaria by complement fixation test by asimplified procedure avoiding the conventional successive freezing andthawing of the blood cells for the extraction of the antigen portion.Thus the time and cost of the process are both reduced materially.

It is a further object of the invention to improve the procedure byearly separation of the parasite mass from the hemoglobin of the bloodcells. This purification is highly advantageous in facilitating theextraction of the mass.

A further object of the invention is to diminish the color of the finalproduct. This is highly important as it makes reading or interpretingthe test much easier and certain.

An added feature of the invention is a great increase in the quantity ofthe antigen obtained from a given source due to the use of an extractantdiffering from the conventional saline solution. Specifically such anextractant may be a phosphate bufier solution or a barbiturate buffersolution.

Other objects of the invention and consequent advantages andimprovements will be evident from the following-detailed description ofthe preferred manner of carrying out my invention.

To obtain a parasite mass for use in obtaining my antigen I infectedmonkeys heavily with malaria through inoculation with the organism knownas Plasmodium Iknowlesi. At the peak of development of the infection themonkeys were killed and the blood collected.

The erythrocytes were laked with distilled water, freeing the parasitesfor further treatment.

The parasites were washed with water and centrifuged to removenon-parasitic material as completely as possible. The parasites werethen dried for subsequent use in the preparation of the antigen. Thisdrying puts the material in form for accurate determination ofquantities for processing. It is otherwise difiicult to measure thequantity of parasites being used or to proportion the extractingmaterial.

The above preparatory routine is mainly preliminary to the principalfeature of my invention and well known except in so far as the lakingstep which is a distinct departure from customary practice.

The parasites prepared as above and in either wet or dry form wereground with M/lO phosphate buffer solution of pH 7.8-8.0 or N/lObarbiturate buffer solution of pH 8.5 prepared by the usual andwell-known method such as is set out on pages 810 and 811 of PracticalPhysiological Chemistry by Hawk and Bergeim, tenth edition 1931. Whilealternate freezing and thawing at room or refrigerator temperatures maybe employed, it is found that the selection of the extracting solutionobviates the need for this routine. The preferred practice as followedwas to allow the preparation to stand at room temperature with frequentstirring for about one hour. Centrifugation yielded a deep brownopalescent supernate. The residue was repeatedly extracted with freshbufier solution until recovery was negligible.

The relative antigenic activity was determined by titration wtih astrongly positive malaria serum. It was found that the bufierpreparations were much more active than the usual saline extracts.

As a specific example of the improved process and to illustrate thecomparative values of this and the conventional saline extraction, two0.1 gram samples of the dried parasites were each ground with 10 cc. ofphosphate buiier and labeled (a) and (b). A third or control sample wasground with 10 cc. of 0.9 percent sodium chloride and labeled (0).

The sample (a) was given the usual treatment by alternate freezing andthawing (four times) then centrifuged, yielding a clear brown supernatewhich was removed and designated P-FT-l. The residue was mixed with afurther 10 cc. of phosphate buifer solution and the alternate freezingand thawing repeated. The supernate from centrifugin was almostcolorless and was designated P-FT-Z.

Two successive supernates P-FT-3 and P-FT- l respectively were obtainedin like manner. Both were practically colorless.

The second sample (1)) was allowed to stand at room temperature withfrequent stirring for about one hour. On centrifugation a deep brownopalescent supernate (P-R-l) was obtained.

The residue was taken up in 10 cc. of buffer and stirred at intervalsfor a second extraction period of about an hour. The pale amberopalescent supernate was designated P-R-Z.

Two additional extractions were made yielding faintly coloredpreparations P-R-3 and P-R-l respectively. The final residue was takenup in 10 cc. of buffer solution, left in the refrigerator overnight andcentrifuged to produce a supernate PR:5.

For purposes of further comparison the sample was frozen and thawed asWas sample (a) but using cc. amounts of 0.9 percent sodium chloridesolution and successive extracts obtained (S-FT-l; S-FT-2; S-FT-3 andS-FT- l respectively) By titration against a strongly positive malariaserum diluted 1:2 the relative antigenic activity of each of the aboveextracts was determined. A known negative serum and anticomplementarycontrols were included. All tests with the negative serum and in theseries containing no serum were negative. The highest dilution of eachantigen giving a 4-plus reaction with the positive serum was recorded.The following table gives the antigen units per cubic centimeterobserved for each extraction in each of the three procedures.

P-FT P-R S-FT Extraction Numbei (buffer) (fligizfggr) (Saline) 7200units by four freezings and thawings in phosphate and 1400 units by fourfreezings and thawings in saline solution.

A further example of the improved process was carried out by treatingfresh or dried parasites with N/lO barbiturate buffer solution (pH 8.5)at room or refrigerator temperatures but otherwise in the sameproportions and conditions as above described.

Either method yielded active antigens containing 320 to 640 units percubic centimeter and additional quantities on repeated extraction.

A highly active antigen was prepared by extracting the wet parasitesobtained from one monkey with barbiturate buifer solution as aboveoutlined, first with 200 cc. and then with 50 cc.

The combined extracts were cleaned of salts by dialysis againstdistilled Water and subsequently concentrated in a semipermeablemembrane at 5 C. to cc. On centrifugation a supernate of 1280 units ofantigen per cc. was obtained.

The antigen produced in the manner herein described is characterized bya marked increase in sensitivity as will be evident from the abovetabulated results of tests. It therefore constitutes a novel product ofmaterially enhanced utility.

Further details relating to the preparation of the antigen and its usein carrying out complement fixation tests for malaria are to be found inthe article on the preparation and properties of antigens fromPlasmodium knowlesi as published in the American Journal of TropicalMedicine, September, 1944, vol. 24, pp. 323-326 by the inventor and D.B. Morrison. In this article (page 324) it is stated that Highly activeantigens may be prepared by treating wet or dried parasites with M/10phosphate bufier of pH LB-8.0.; and (page 325) with N/lO barbituratebuffer pH 8.5.

From the above description it will be evident that I have provided amaterial and distinct improvement in the process of preparing an antigenfor complement fixation tests for malaria. The process is marked bysimplification in routine with consequent saving in time and cost. Thefinal product is improved in color and renders making the tests moreeasy and certain. Finally the quantity of antigen recovered has beensubstantially increased and the antigen found to have greatersensitivity or activity. Other incidental advantages are present and theinvention is clearly subject to variation in minor details and factorswithin the scope of the appended claims.

This application is a division of Serial No. 616,959, filed September17, 1945, which parent application covers the phosphate buffer specieswhereas the present application covers the barbiturate buffer species.

What is claimed is:

1. The process of preparing an antigen for complement fixation tests formalaria from parasitized animal blood which consists in laking the bloodcells, grinding the parasite mass in N/10 barbiturate buffer solution ofpH 8.5, allowing the mixture to stand for approximately one hour withfrequent agitation and separating the antigen.

2. The process of preparing an antigen for complement fixation tests formalaria from the blood of animals infected with Plasmodz'um knowlesi,which consists in laking the blood cells, grinding the parasite mass inN/10 barbiturate buffer solution of pH 8.5, allowing the mixture tostand for approximately one hour with frequent agitation and separatingthe antigen.

3. The process of preparing an antigen for complement fixation tests formalaria from the blood of animals infected with Plasmodium knowlesz,which consists in laking the blood cells, grinding the parasite mass inN/10 barbiturate buffer solution of pH 8.5, allowing the mixture tostand for approximately one hour with frequent agitation, centrifugingto separate supernatant antigen, repeating the extraction with freshbuffer solution and combining the several supernates.

4. The process of preparing an antigen for complement fixation tests formalaria from the blood of animals infected with Plasmodium 5 knowlesi,which consists in grinding the parasitecontaining mass in N/ 10barbiturate buffer solution of pH 8.5, allowing the mixture to stand forapproximately one hour with frequent agitation and separating theantigen.

5. An antigen for complement fixation tests for malaria consisting ofthe extract in N/ 10 barbiturate buffer solution of pH 8.5 of theparasites from the blood of an animal infected with Plasmodiumknowlesz'.

ANNA DEAN DULANEY.

REFERENCES CITED The following references are of record in the file ofthis patent:

Chemical Abstract, vol. 36, 1942, columns 4887, 4888; ComplementFixation in Human Malaria, by Dulaney et al. and Buffer PrecipitationTests for Malaria, by E. K. Wolff, (Copy in Patent Ofiice Library.)

